ORIGINAL RESEARCH published: 22 February 2016 doi: 10.3389/fmicb.2016.00127 Viral Outbreak in Corals Associated with an In Situ Bleaching Event: Atypical Herpes-Like Viruses and a New Megavirus Infecting Symbiodinium Adrienne M. S. Correa1,2, Tracy D. Ainsworth3, Stephanie M. Rosales1, Andrew R. Thurber4, Christopher R. Butler1,5 and Rebecca L. Vega Thurber1* 1 Department of Microbiology, Oregon State University, Corvallis, OR, USA, 2 BioSciences at Rice, Rice University, Houston, TX, USA, 3 ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD, Australia, 4 College of Earth, Ocean, and Atmospheric Sciences, Oregon State University, Corvallis, OR, USA, 5 Department of Viticulture and Enology, University of California at Davis, Davis, CA, USA Previous studies of coral viruses have employed either microscopy or metagenomics, Edited by: but few have attempted to comprehensively link the presence of a virus-like particle Ian Hewson, (VLP) to a genomic sequence. We conducted transmission electron microscopy imaging Cornell University, USA and virome analysis in tandem to characterize the most conspicuous viral types found Reviewed by: within the dominant Pacific reef-building coral genus Acropora. Collections for this Fabiano Thompson, Federal University of Rio de Janeiro, study inadvertently captured what we interpret as a natural outbreak of viral infection Brazil driven by aerial exposure of the reef flat coincident with heavy rainfall and concomitant Stéphan Jacquet, Institut National de la Recherche mass bleaching. All experimental corals in this study had high titers of viral particles. Agronomique, France Three of the dominant VLPs identified were observed in all tissue layers and budding Mya Breitbart, out from the epidermis, including viruses that were ∼70, ∼120, and ∼150 nm in University of South Florida, USA diameter; these VLPs all contained electron dense cores. These morphological traits are *Correspondence: Rebecca L. Vega Thurber reminiscent of retroviruses, herpesviruses, and nucleocytoplasmic large DNA viruses [email protected] (NCLDVs), respectively. Some 300–500 nm megavirus-like VLPs also were observed Specialty section: within and associated with dinoflagellate algal endosymbiont (Symbiodinium) cells. This article was submitted to Abundant sequence similarities to a gammaretrovirus, herpesviruses, and members Aquatic Microbiology, of the NCLDVs, based on a virome generated from five Acropora aspera colonies, a section of the journal Frontiers in Microbiology corroborated these morphology-based identifications. Additionally sequence similarities Received: 11 September 2015 to two diagnostic genes, a MutS and (based on re-annotation of sequences from Accepted: 25 January 2016 another study) a DNA polymerase B gene, most closely resembled Pyramimonas Published: 22 February 2016 orientalis virus, demonstrating the association of a cosmopolitan megavirus with Citation: Correa AMS, Ainsworth TD, Symbiodinium. We also identified several other virus-like particles in host tissues, Rosales SM, Thurber AR, Butler CR along with sequences phylogenetically similar to circoviruses, phages, and filamentous and Vega Thurber RL (2016) Viral viruses. This study suggests that viral outbreaks may be a common but previously Outbreak in Corals Associated with an In Situ Bleaching Event: Atypical undocumented component of natural bleaching events, particularly following repeated Herpes-Like Viruses and a New episodes of multiple environmental stressors. Megavirus Infecting Symbiodinium. Front. Microbiol. 7:127. Keywords: virome, tropical coral reef, virus-like particle (VLP), herpesvirus, megavirus, nucleocytoplasmic large doi: 10.3389/fmicb.2016.00127 DNA virus (NCLDV) Frontiers in Microbiology | www.frontiersin.org 1 February 2016 | Volume 7 | Article 127 Correa et al. Natural Viral Outbreak in Acroporid Corals INTRODUCTION this issue (e.g., Patten et al., 2008). The NCLDV group includes the Phycodnaviridae, Iridoviridae, Poxviridae, Mimiviridae,and Viruses (phages and eukaryotic viruses) are abundant and diverse Ascoviridae, as well as the recently described giant viruses, residents of stony coral colonies (reviewed in Vega Thurber marseillevirus and lausannevirus (Iyer et al., 2006; Yamada, and Correa, 2011). These viruses likely play multiple, parasitic 2011; Yutin and Koonin, 2012). VLPs that are within the and commensal roles in the health of coral reefs (e.g., Wilson cytoplasm, larger than 120 nm, and generally icosahedral in et al., 2005; van Oppen et al., 2009; Rosenberg and Zilber- shape are often interpreted as NCLDV-like but several exceptions Rosenberg, 2014; Bettarel et al., 2015; Weynberg et al., 2015). to this rule remain, such as the poxviruses, and pandoravirus, Research interrogating the impact of viruses on colony fitness and which are NCLDVs that exhibit very different morphological survival under different environmental contexts is of particular characteristics. importance, given anthropogenic climate forcing and other Genomic and proteomic-based studies have identified impacts (van Oppen et al., 2009). For example, abiotic conditions patterns in the diversity and abundance of genomic sequences that stress coral colonies, such as elevated seawater temperatures similar to described viruses within healthy and diseased tropical or UV exposure, may trigger viral infections that contribute corals (Wegley et al., 2007; Marhaver et al., 2008; Vega Thurber to coral bleaching and disease (Vega Thurber and Correa, et al., 2008; Littman et al., 2011; Weynberg et al., 2014; Wood- 2011; Wilson, 2011; Lawrence et al., 2015). Identifying potential Charlson et al., 2015)andculturedSymbiodinium (Wilson et al., mechanisms of coral reef decline is increasingly important given 2005; Claverie et al., 2009; Weston et al., 2012; Correa et al., accelerations in this process during recent decades (e.g., Gardner 2013; Lawrence et al., 2014; Nguyen-Kim et al., 2014; Soffer et al., 2003; De’Ath et al., 2012), and the current global mass et al., 2014a,b), as well as in cold water corals (Maier et al., bleaching event1. 2011; Rosario et al., 2015). For example, a strong correlation Although the field of coral virology remains in its infancy, between specific viral markers in bleached, diseased, and healthy several groups have applied microscopy or genomics to examine Orbicella corals was used to establish a role for small circular the diversity and roles of viruses in coral holobionts. Microscopy ssDNA viruses (SCSDVs) in white plague disease (Soffer et al., studies have presented evidence that virus-like particles (VLPs) 2014a). Although this work was somewhat substantiated by a are present in all tissue layers of apparently healthy and diseased TEM study on another coral white disease (Pollock et al., 2014), corals: the gastrodermis, mesoglea, and epidermis, as well as viral metagenome studies often contain sequence similarities to in the coral surface microlayer (CSM; e.g., Patten et al., 2008; many viral groups, most of which have not been corroborated by Leruste et al., 2012; Bettarel et al., 2013; Nguyen-Kim et al., microscopy-based studies. For example, numerous studies have 2014; Pollock et al., 2014). The physical structure of VLPs also found sequences similar to mimiviruses and baculoviruses, and has been examined within cultured Symbiodinium (Wilson et al., yet no TEM study has confirmed these annotations (Claverie 2001; Lohr et al., 2007; Lawrence et al., 2014). Some of these et al., 2009; Sharma et al., 2014; Wood-Charlson et al., 2015). This observed VLPs likely represent particles produced during the lytic is likely due to several issues. A single sequence read or contig replication phase of previously latent or endogenous infections may have significant similarity to multiple viral groups because of the coral animal, its dinoflagellate algae, or its microbiota many related viruses share some gene homology. Alternatively, (Patten et al., 2008; Wilson, 2011). Davy and Patten (2007), reads or contigs may have only a few sequence similarities with for example, were able to distinguish 17 sub-groups of VLPs relatively high associated e-values. It can also be difficult to rule associated with the CSM of four species of Australian corals based out contamination as a source of error for sequence similarities on morphological similarities. The role of each of these groups represented by few reads within a metagenome, or for reads of viruses is uncertain, especially considering that the density of similar to cosmopolitan or host-associated viral remnants (e.g., some VLPs within the CSM is relatively low. However, in other retrotransposons, retroelements). Thus, ambiguity remains when cases, transmission electron microscopy has revealed structures metagenomics is the sole approach applied to characterize the within corals that are highly indicative of massive viral infection viral consortia associated with corals. (e.g., crystalline arrays, viral factories; Lawrence et al., 2014). A comparative analysis of several metagenome studies Yet standalone transmission electron microscopy (TEM) recently addressed some of these challenges and cataloged a images can pose interpretive challenges. A set of TEM images cosmopolitan set of viruses in corals and their symbionts (Wood- may contain VLPs that present only some of the diagnostic Charlson et al., 2015). This meta-analysis showed that, based on morphological characteristics of a viral group, or characteristics presence in >90% of 35 surveyed metagenomes, coral